Surface Enhanced Transmission Mode Desorption Electrospray Ionization for Metabolite Profiling from Dried Blood Spots This application addresses broad Challenge Area (03) Biomarker Discovery and Validation, and specific Challenge Topics, 03-AG-101: Novel assays for dried blood spots and 03-AG-108: Developing high-throughput biomarker assays from finger-stick dried blood spots. We propose a new surface-enhanced mass spectrometric strategy for sensitive, high throughput analysis of metabolites in dried blood spots. Metabolomics, which encompasses the study of all small molecules found in tissues, cells, or biological fluids, presents a compelling frontier for exploration in a variety of health applications, including toxicology, pharmacology, physiology, the search for biomarkers of disease, and unraveling biochemical mechanisms of disease. The potential to monitor health status and the opportunity for early diagnosis of disease based on mapping biomarkers such as proteins, lipids, and metabolites has led to an increasing emphasis on the development of methodologies for rapid, high throughput identification of biological molecules in complex mixtures with high sensitivity and minimal sample consumption. In particular, the development of high throughput methods for rapid screening of mixtures to extract key information about targeted subsets of molecules remains a high priority in the metabolomics arena. In the context of the advances in mass spectrometric methods for metabolomics over the past decade, issues related to high throughput capacity, minimization of sample processing, enhanced selectivity, and high sensitivity remain top priorities. These analytical challenges are addressed in this proposal via the development of surface-enhanced desorption ionization tandem mass spectrometry for metabolomic profiling from dried blood spots. The proposed methodology combines ambient ionization (desorption electrospray ionization, DESI, or direct analysis in real time, DART), a new class of robust ionization methods, with selective surface chemistry to create a tunable extraction/desorption/ionization platform that allows the capture, enrichment, and mass spectrometric analysis of targeted compounds from dried blood spots without the need for extensive sample processing or chromatographic separation. The analysis of dried blood spots offers a compelling matrix for extracting health- related information because of the large range of compounds present in blood, the relative ease of sample collection (relying on a finger stick as opposed to more invasive venipuncture), the far more simplified sample processing, the stability of the sample constituents when stored under dry conditions, the reduction of sample volume, the ease of storage, and the minimal biohazard risk compared to other biological matrices. The proposed work includes four key objectives: 1) Development and application of surface-enhancement strategies based on modification of meshes with selective capture materials 2) Determination of analytical figures-of-merit for transmission mode DESI-MS and DART- MS methods, including implementation of quantitative analysis protocols 3) Comparison and assessment of both TMDESI-MS and TMDART-MS methods for high throughput profiling of metabolites 4) Evaluation, optimization, and validation of the surface-enhanced transmission mode DESI- and DART-MS methods for direct analysis of blood spots PUBLIC HEALTH RELEVANCE: Metabolomics, which encompasses the study of all small molecules found in tissues, cells, or biological fluids, presents a compelling frontier for exploration in a variety of health applications, including toxicology, pharmacology, physiology, the search for biomarkers of disease, and unraveling biochemical mechanisms of disease. We propose a new surface-enhanced mass spectrometric strategy for sensitive, high throughput analysis of metabolites in dried blood spots.